Immunocytochemistry on free-floating sections of rat brain using microwave irradiation during the incubation in the primary antiserum: light and electron microscopy.
We studied the effects of microwave irradiation during the incubation of free-floating brain sections with primary antibodies against gamma-aminobutyric acid (GABA), enkephalin and vasopressin. Vibratome sections of perfusion-fixed rat brain were incubated: (a) overnight at room temperature (20-22 degrees C), (b) during various periods of time under microwave irradiation, such that the induced temperatures did not exceed 10 degrees C, (c) same as (b) but with induced temperatures not exceeding 40 degrees C, (d) without microwave irradiation, at 4-10 degrees C (temperature control for (b)), (e) same as (d) but at 40 degrees C (temperature control for (c)). During the incubation-irradiation we continuously monitored the temperature and controlled it by cooling and by manipulating the energy output of the magnetron. The peroxidase immunocytochemical procedure was completed using for all sections the same incubation parameters. Selected GABA-immunoreacted sections were examined in the electron microscope. Incubation at 10 degrees C in the primary antiserum as short as 30 min, with or without microwave irradiation, already results in (weak) binding of the antibodies to immunoreactive structures. One or 2 h of incubation in the primary antiserum in the microwave oven at 40 degrees C or at the same temperature outside the microwave oven results in excellent staining of GABA-immunoreactive structures and of good staining of enkephalin- or vasopressin-immunoreactive structures. The ultrastructural details were much better preserved in incubated-irradiated sections than in sections incubated overnight and only slightly less preserved than in the other control sections. There is no improved penetration of the antibodies into the sections. We conclude that by using microwave technology or by raising the temperature of the incubation medium, the time of incubation, at least in these antisera, can be shortened drastically, whereas the ultrastructural details remain well preserved.
Wouterlood
F G
FG
Department of Anatomy, Vrije Universiteit, Amsterdam, The Netherlands.
Boon
M E
ME
Kok
L P
LP
D000818:Animals; D001921:Brain / Q000033:anatomy & histology* / Q000648:ultrastructure; D004745:Enkephalins / Q000276:immunology; D005260:Female; D007106:Immune Sera / Q000276:immunology*; D007124:Immunoenzyme Techniques; D016253:Microscopy, Immunoelectron; D008872:Microwaves; D051381:Rats; D011919:Rats, Inbred Strains; D013048:Specimen Handling; D013696:Temperature; D014667:Vasopressins / Q000276:immunology; D005680:gamma-Aminobutyric Acid / Q000276:immunology
D004745:Enkephalins; D007106:Immune Sera; D014667:Vasopressins; D005680:gamma-Aminobutyric Acid